Rhesus monkey tryptophan hydroxylase-2 coding region haplotypes affect mRNA stability
Rhesus monkey tryptophan hydroxylase-2 coding region haplotypes affect mRNA stability
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DOI：
10.1016/j.neuroscience.2008.05.050
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发表时间：
2008-08-13
期刊：
影响因子：
3.3
通讯作者：
Miller, G. M.
中科院分区：
文献类型：
--
作者：
Chen, G. -L.;Miller, G. M.
色氨酸羟化酶-2（TPH 2）合成神经元5-HT，其遗传变异与许多行为特征和精神疾病相关。本研究旨在探讨恒河猴TPH 2（mTPH 2）中两个非同义单核苷酸多态性（SNP）（C74 A和G223 A）的功能意义。将4种单倍型的mTPh 2基因克隆到pcDNA3.1中，并稳定转染PC 12细胞。采用实时荧光定量PCR和Western blot检测mTPH 2 mRNA和蛋白水平，酶联免疫吸附试验（ELISA）检测细胞内5-HT含量。与野生型C-G单倍型相比，变异型A-A单倍型的mTPH 2 mRNA和蛋白表达水平显著升高，5-HT产量显著增加，而其他两种变异型（C-A和A-G）在PC 12细胞中稳定表达时，5-HT产量也显著高于C-G单倍型。预测C74 A和G223 A都改变mRNA二级结构，并且mRNA稳定性的分析表明，在稳定的PC 12和瞬时HEK-293细胞中，野生型C-G单倍型mRNA比突变型mTPH 2单倍型mRNA降解得更快。这项研究表明，mTPH 2中的非同义SNP可以影响mRNA的稳定性。我们的研究结果提供了一个额外的机制，非同义SNP影响TPH 2功能，并进一步了解TPH 2基因表达调控。(C)2008年IBRO。由爱思唯尔有限公司出版。保留所有权利。
Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal 5-HT and its genetic variance is associated with numerous behavioral traits and psychiatric disorders. This study characterized the functional significance of two nonsynonymous single nucleotide polymorphisms (SNPs) (C74A and G223A) in rhesus monkey TPH2 (mTPH2). Four haplotypes of mTPh2 were cloned into pcDNA3.1 and stably transfected into PC12 cells. The levels of mTPH2 mRNA and protein were assessed by quantitative real-time PCR and Western blot, respectively, while the intracellular 5-HT was measured by enzyme-linked immunosorbent assay (ELISA). The variant A-A haplotype showed significantly higher levels of mTPH2 mRNA and protein, as well as significantly higher 5-HT production than the wild-type C-G haplotype, while the other two variant haplotypes (C-A and A-G) also tended to produce more 5-HT than C-G haplotype when stably expressed in PC12 cells. Both C74A and G223A were predicted to change mRNA secondary structure, and analysis of the mRNA stability showed that the wild-type C-G haplotype mRNA degrades more quickly than mRNAs of the mutant mTPH2 haplotypes in both stable PC12 and transient HEK-293 cells. This study demonstrates that nonsynonymous SNPs in mTPH2 can affect mRNA stability. Our findings provide an additional mechanism by which nonsynonymous SNPs affect TPH2 function, and further our understanding of TPH2 gene expression regulation. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.